HIV latently infected cells are very rare in HIV+ individuals on suppressive anti-retroviral therapy (ART). To target these latently infected cells without latency-reactivation, it is critical to identify specific cell surface targets associated wth the latent infection. Compelling reports from studying proteins such as CD2, PD-1, LAG-3 and TIGIT suggest that there is a distinct profile of cell surface proteins for enrichment of latently infected cells ex vivo. The specific changes of these proteins during the establishment of latent infection remain to be elucidated. Interestingly, all of the above proteins are glycosylated (glycoproteins). It is estimated that there are over 1,000 cell surface glycoproteins that form a complex and dynamic cell surface phenotype. Interrogating the cell surface glycoproteome will maximize the chances to identify the specific cell surface glycoproteins to target latently infecte cells. Toward this goal, we have developed integrated glycoproteomics that can quantify the glycoproteome using chemical-enzymatic approaches for glycoprotein isolation followed by quantitative analysis using state-of-the-art mass spectrometry (MS). Our innovative method acquires a direct readout from the cell surface glycoproteome. The workflow is coupled with high throughput screen/validation assays using parallel reaction monitoring based mass spectrometry (PRM-MS) to monitor the otherwise complex surface glycoproteome of latently infected cells. We therefore propose to screen the cell surface glycoproteins in an in vitro-generated latently infected primary cell model to reveal latency- associated cell surface glycoproteins using glycoproteomics and PRM-MS in the R21 phase and target latently infected cells ex vivo using antibodies in the R33 phase. In the R21 phase, we will aim to discover the dynamic change of the glycoproteome during establishment of an in vitro-generated latently infected primary cell model (aim 1). The latency-associated glycoproteins will be validated using parallel reaction monitoring mass spectrometry (PRM-MS), a targeted mass spectrometry technique that can validate a large number of changed glycoproteins (aim 2). The analytical methods are highly innovative and have the capability to uncover the specific glycoprotein changes from thousands of cell surface glycoproteins for targeting latently infected cells. The R33 phase will be undertaken only if the well-defined milestones are achieved. We propose three studies to determine the roles of specific glycoprotein changes on latently infected cell surface ex vivo (aim 3). (1) The correlation of glycoproteins to latent infection will be determine to uncover latency-associated glycoproteins. (2) Glycoprotein-specific antibody will be used to capture latently infected cells. (3) A panel of glycoproteins will be selected to improve specificiy and/or coverage of targeting latently infected cells. The overall goal of this study is to determin the specific cell surface glycoprotein(s), which enable targeting the rare latently infected cells x vivo without the need to reactivate HIV gene expression.